Many bacteria secrete protein toxins which form pores in host membranes as part of their infective cycle. Examples include Corynebacterium diphtheriae, Pseudomonas aeruginosa, Bacillus anthracis, and Staphylococcus aureus. In many cases these secreted toxins are necessary for or are associated with bacterial virulence. For example, Corynebacterium diphtheriae is only virulent if it secretes diphtheria toxin. (see Pappenheimer, A. M., Jr., Annu. Rev. Biochem. 46, 69-94 (1977)). In addition, these toxins often cause tissue damage.
Diphtheria toxin is illustrative of the membrane pore forming bacterial toxins. This toxin is secreted as a single polypeptide (Mr 58,348), but can be readily cleaved by a furin-like protease into two subunits, A (Mr 21,167) and B (Mr 37,199), which are held together by a disulfide bond. Diphtheria toxin enters cells by receptor-mediated endocytosis, and, once internalized, the acidification of the endosomal lumen triggers a pH dependent conformational change which causes the toxin to become hydrophobic and penetrate the endosomal membrane. See Gordon, V. M., et al., Infect. Immun. 63, 82-87 (1995) and London, E., Biochim. Biophys. Acta 1113, 25-51 (1992). After reduction of the disulfide bond holding the A and B subunits together, the A chain is translocated across the membrane bilayer into the cytoplasm where it catalyzes the ADP-ribosylation of elongation factor 2, inhibiting protein synthesis and causing cell death. See, for example, Collier, R. J. in ADP Ribosylation reactions: Biology and Medicine (Hayashi, O. and Ueda, K., eds.) pp. 573-592 (1982), Academic Press, New York.
The mechanism of A chain translocation across membranes involves pore formation. For example, see Mindell, J. A., et al. J. Memb. Biol. 137, 45-57 (1994); Jiang, G.-S., et al. J.Biol.Chem. 264, 13424-13429 (1989); Kagan, B. L., et al. Proc. Natl.Acad.Sci. USA 78, 4950-4954 (1981); and Donovan, J. J., et al. Proc.Natl.Acad. Sci. USA 78, 172-176 (1981). Significantly, using mutant toxins, it has been shown that membrane pore formation by the toxin is associated with its translocation and toxicity. See Falnes, P. O., et al. J.Biol.Chem. 267, 12284-12290 (1992); and Silverman, J. A., et al. J.Biol.Chem. 269, 22524-22532 (1994).
While bacterial infections are usually treated with antibiotics which destroy bacteria and/or interfere with bacterial growth, resistance to standard antibiotics is an increasingly serious problem. Moreover, for bacterial infections involving toxin production, using antibiotics after symptoms have manifested will often be too late because considerable toxin production will already have occurred, and antibiotics are not effective against toxins.
In cases where toxin production has already occurred, antitoxins have been used. However, since these antitoxins are antibodies, they only bind to toxin in the circulatory system and cannot act on toxins which have already been internalized into cells. Antitoxins currently used are animal products in which contamination by pathogens (including virus) can occur. Moreover, antitoxins are unstable, have a limited shelf life, and must be stored under refrigeration. Antitoxins must also be administered by injection. These disadvantages have greatly limited the use of antitoxins and there is a need for new therapeutic approaches to this problem.
Cibacron dyes have been shown to bind to the catalytic (enzymatic) site of diphtheria toxin at neutral pH, i.e. when the toxin is not in the conformation that inserts in membranes. See, for example, Antoni, G., et al., Experiential 39, 885-886 (1983); Rambelli, F., et al., Bioscience Reports, 7, 737-743 (1987). Diphtheria toxin enzymatic activity occurs at a step in the infective cycle after pore formation. There is no suggestion that Cibacron dyes or any other molecules affect pore formation.
Cibacron blue, and other molecules with similar chemical moieties, have been shown to affect diphtheria toxin translocation across Vero cell membranes. For example, Moskaug, J. O., et al., J.Biol.Chem. 263, 2518-2525 (1988), found that DIDS, an inhibitor of anion transport, blocks the translocation of the A fragment of diphtheria toxin across cell membranes and reduces the insertion of a 25 kDa fragment of the protein into cell membranes. From these results, the authors concluded that either anion transport is necessary for toxin translocation across the membrane or that an anion transporter might be a receptor for the toxin. No suggestion that DIDS affects pore formation was made. An article by Falnes, P. .O slashed.., and Olsnes, S., J.Biol.Chem. 270, 20787-20793 (1995), discloses DIDS, NEM, and Cibacron blue as inhibitors of diphtheria toxin translocation across Vero cell membranes. This article teaches that Cibacron Blue and DIDS disrupt translocation by inhibiting anion transport. NEM is hypothesized to act by quenching cellular reducing agents. Inhibition of pore formation is not suggested.
None of the references cited above teach or suggest that the molecules described therein, or any other molecules, inhibit membrane pore formation by protein toxins. Nor do these references teach or suggest methods for developing pharmaceuticals whose mechanism of action is the inhibition of membrane pore formation by protein toxins.
In view of the above considerations, it is clear that methods for reducing the effects of toxins and of treating bacteria harboring toxins have been limited. Accordingly, it is one of the purposes of the present invention to overcome these limitations in treatment of infections where the mechanism of virulence involves membrane pore formation by protein toxins. The invention provides methods for obtaining agents that are effective at inhibiting pore formation in membranes by protein toxins.